RU2735291C1 - Vaccine strain of influenza virus a/17/brisbane/2018/8175 (h1n1) pdm09 for production of live influenza intranasal vaccine for adults and children - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Invention is a strain of virus A/17/Brisbane (2018/8175 (H1N1) pdm09 – reassortant, produced by crossing epidemic virus A/Brisbane/02/2018 (H1N1) pdm09 with cold-adapted temperature-sensitive virus A/Leningrad/134/17/57 (H2N2) – an attenuation donor harmless for humans.

EFFECT: invention can be used for prevention of influenza prevalence among adults and children with the help of live influenza intranasal vaccine of strain of influenza A/17/Brisbane/2018/8175 (H1N1) pdm09 virus.

1 cl, 5 tbl

Description

The invention relates to medical virology and can be used in healthcare for the prevention of the incidence of influenza among adults and children using a live influenza intranasal vaccine from a strain of influenza A virus, the Orthomyxoviridae family, the genus Influenzavirus A, A / 17 / Brisbane / 2018/8175 (H1N1) pdm09.

In February 2019, WHO (World Health Organization) made recommendations to replace the H1N1pdm09 component in influenza vaccines for the Northern Hemisphere for the 2018-2019 epidemic season. on A / Brisbane / 02/2018 (H1N1) dpm09-like viruses [WHO. Recommended composition of influenza virus vaccines for use in the 2019-2020 northern hemisphere influenza season. 21 February 2019. https://www.who.int/influenza/vaccines/virus/recommendations/2019_20_ north / en /].

As a result of the appearance in the circulation of a new highly epidemic strain of influenza virus, A / Brisbane / 02/2018 (H1N1) pdm09, the known vaccine strain A / 17 / New York / 15/5364 (H1N1) pdm09 is a prototype [Dubrovina I.A., I. V. Kiseleva, L. G. Rudenko Vaccine strain of influenza virus A / 17 / New York / 15/5364 (H1N1) pdm09 for the production of live influenza intranasal vaccine for adults and children. RF patent No. 2653388. Posted on 08.05.2018, Bull. No. 13] - has lost its relevance.

The problem to be solved by the claimed invention is to obtain an antigenically relevant A / Brisbane / 02/2018 (H1N1) pdm09-like vaccine strain of live influenza vaccine for adults and for children based on a cold-adapted attenuation donor A / Leningrad / 134/17/57 (H2N2) and the new epidemic virus A / Brisbane / 02/2018 (H1N1) pdm09.

Currently used strains for live influenza vaccines are obtained by the method of genetic reassortment of epidemically relevant viruses with cold-adapted strains - attenuation donors [Alexandrova G.I. Application of the method of genetic recombination to obtain vaccine strains of the influenza virus. Virology issues. 1977. No. 4. S. 387-395].

Attenuation donor A / Leningrad / 134/17/57 (H2N2) is a cold-adapted (with a ) and temperature-sensitive (ts) strain of the influenza virus, approved by the Ministry of Health of the Russian Federation for obtaining harmless live intranasal vaccines for adults and children [Alexandrova G.I. New in epidemiology and prevention of viral infections. L., 1968. S. 66-83].

The purpose of reassortment is to obtain a strain with a vaccine genome formula of 6: 2. The genes encoding the surface proteins of the influenza virus hemagglutinin (HA) and neuraminidase (NA) are inherited from an antigenically relevant circulating epidemic strain, and six genes encoding internal and non-structural proteins (PB2, PB1, PA, NP, M, NS) are inherited from a harmless donor attenuation. Based on the attenuation donor A / Leningrad / 134/17/57 (H2N2), the vaccine strain A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 was created.

Obtaining a vaccine strain. The vaccine strain A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 was obtained by the method of genetic reassortment of the epidemic virus A / Brisbane / 02/2018 (H1N1) pdm09 with the attenuation donor A / Leningrad / 134/17/57 (H2N2) by simultaneous infection developing chicken embryos (ECE) with a mixture of parental viruses in equivalent infectious doses, followed by selection of clones with desired properties at an incubation temperature lowered to 26 ° C in the presence of an antiserum to an attenuation donor. The clones were additionally purified by three consecutive clonings by the limiting dilution method in the presence of antisera to the attenuation donor at a reduced (26 ° C) and optimal (32 ° C) incubation temperatures. A pure clone was tested for phenotypic characteristics (ts, with a -phenotype) and according to the genome formula for compliance with the requirements for a reassortant vaccine strain.

Antigenic characteristics of the vaccine strain.

The study of the antigenic properties of the vaccine strain A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 showed that:

- the surface protein of the vaccine strain, hemagglutinin (HA), responsible for the antigenic specificity, is antigenically identical to the epidemic virus A / Brisbane / 02/2018 (H1N1) pdm09 in RTGA with homologous serum, since the interaction indicators of the reassortant A / 17 / Brisbane / 2018/8175 ( H1N1) pdm09 and the parental strain A / Brisbane / 02/2018 (H1N1) pdm09 with rat antiserum obtained against the eaidemic virus A / Brisbane / 02/2018 (H1N1) pdm09, completely coincided (Table 1). Correspondence of the protein sequence of the vaccine reassortant A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 and the parental virus A / Brisbane / 02/2018 (H1N1) pdm09 was confirmed by sequencing of the corresponding genome segment;

- the correspondence of the second responsible for the antigenic specificity of the surface protein of the vaccine strain - neuraminidase (NA) - was confirmed by determining the genetic sequence of the bgo genome segment, according to which it corresponds to the 6th segment of the genome of the virus A / Brisbane / 02/2018 (H1N1) pdm09.

Partial DNA sequencing of gene copies was used to analyze the genome composition of the obtained reassortants [Matyushenko V. et al. Genotyping assay for differentiation of wild-type and vaccine viruses in subjects immunized with live attenuated influenza vaccine. PLoSOne. 2017. Vol. 12 No. 7. P.e0180497. doi: 10.1371 / journal.pone.0180497].

The 6: 2 genome formula meets the requirements for live influenza vaccine strains: the genes encoding the surface proteins hemagglutinin (HA) and neuraminidase (NA) belong to the pandemic parental virus A / Brisbane / 02/2018 (H1N1) pdm09, and the genes encoding internal proteins (PB2, PB1, PA, NP, M, NS), - to the attenuation donor A / Leningrad / 134/17/57 (H2N2). The results of the analysis of all genes of the vaccine strain A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 are presented in table. 2.

The data obtained during the initial selection of the vaccine candidate was confirmed by full sequencing of its genome and showed that the vaccine strain A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 inherited all 6 genes encoding internal and non-structural proteins from the attenuation donor A / Leningrad / 134/17/57 (H2N2) (Table 2).

Thus, the complete genome sequencing of the A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 strain confirmed its vaccine genome formula.

Genetic stability of coding mutations. The genetic stability of the vaccine strain A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 was studied by comparing the persistence of coding mutations in internal genes before and after five-fold passaging of the vaccine strain prepared on the basis of the attenuation donor A / Leningrad / 134/17/57 in chicken embryos using the pyrosequencing method. For this analysis, specific primers were developed to assess the presence of these mutations using the PyroMark Q24 pyrosequenator. Primers were developed using the PSQ AssayDesign computer program. The obtained pyrosequencing data confirmed the presence of all coding nucleotide substitutions, characterized for the attenuation donor A / Leningrad / 134/17/57 (H2N2) as responsible for its attenuation, in the internal genes of the reassortant strain A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 before and after passaging it five times (Table 3).

All of the above confirms the high genetic stability of the vaccine strain A / 17 / Brisbane / 2018/8175 (H1N 1) pdm09.

The assessment of the phenotypic properties of the vaccine strain A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 was carried out by its parallel titration in developing chicken embryos at different incubation temperatures. Vaccine strain A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 has a phenotype identical to the phenotype of the attenuation donor A / Leningrad / 134/17/57 (H2N2). It was found that the vaccine virus is temperature-sensitive (ts phenotype) - its infectious activity at 40 ° C was less than ≤1.2 log ₁₀ EID ₅₀ / ml, and cold-adapted (with a phenotype) - infectious activity at an incubation temperature lowered to 26 ° C was 5.4log ₁₀ EID ₅₀ / ml, which indicates its harmlessness to humans, since according to these parameters it is identical to the attenuation donor A / Leningrad / 134/17/57 (H2N2). All results of phenotypic analysis are presented in table. 4.

Harmless to mice. Preclinical studies of acute toxicity of the vaccine strain A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 were carried out on white outbred mussels in accordance with the Guidelines for conducting preclinical studies of drugs [Guidelines for conducting preclinical studies of drugs (Immunobiological drugs); Part two. - M .: Grif and K, 2012. - 536 p] and with the "Rules of Good Laboratory Practice" [Rules of Good Laboratory Practice. Order of the Ministry of Health of the Russian Federation No. 199n dated 01.04. 2016. https://rg.ru/2016/09/02/minzdrav-prikaz 199-site-dok.html].

The mice were injected once with one human dose (0.5 ml) of the vaccine virus intraperitoneally at a titer of 7.0 lg EID ₅₀ / ml. The control animals were injected intraperitoneally with phosphate buffered saline (PBS). Every day during the entire study (7 days), the general condition of each animal was monitored.

Physiological research data (mobility, behavioral reactions, kinetics of body weight) showed that intraperitoneal administration of the vaccine strain did not cause death of experimental animals (Table 5) and did not lead to a change in their appearance, behavior, did not affect their food and water consumption. which indicates the harmlessness of the vaccine preparation.

Conclusion

As a result of the preclinical studies, it was established that the claimed vaccine strain of live influenza vaccine A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 is characterized by a combination of useful traits required by the vaccine strain: antigenic specificity of the epidemic virus A / Brisbane / 02/2018 (H1N1) pdm09, genome structure 6: 2, optimal for reassortant vaccine strains, as well as the characteristic donor attenuation temperature sensitivity and cold adaptation and harmlessness for laboratory animals, which correlates with attenuation for humans. A sample passport for the vaccine strain A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 is attached.

Thus, the vaccine strain A / 17 / Brisbane / 2018/8175 (H1N1) pdm09, in terms of the main biological properties studied in preclinical in vitro and in vivo experiments, meets the requirements for vaccine strains of the Pharmacopoeia Article (FSP: P N003224 / 01- 270313) on Ultravac ^® , live allantoic influenza vaccine for intranasal use for adults and children.

The resulting strain A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 was deposited on 12.07.2019 in the State Collection of Viruses of the Institute of Virology named after I. DI. Ivanovsky Federal State Budgetary Institution “NITsEM named after N.F. Gamaleya "of the Ministry of Health of Russia under No. 2931.

CHARACTERISTIC OF THE STRAIN

The infectious activity of the A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 strain during reproduction in developing chicken embryos at 32 ° C for 48 hours is 8.4 log ₁₀ EID ₅₀ / ml. Hemagglutinating activity - 1: 256.

The strain shows genetic stability of phenotypic traits after 5 passages in chick embryos (when using large infectious doses).

A useful property of the vaccine strain of the influenza A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 virus is its suitability for the production of a live influenza vaccine. The inventive vaccine strain of influenza A / 17 / Brisbane / 2018/8175 (H1N1) pdm09 can be used for the prevention of influenza in both adults and children from the age of three.

Claims (1)

Influenza virus strain A / 17 / Brisbane / 2018/8175 (H1N1) pdm09, deposited in the State Collection of Viruses of the Institute of Virology named after DI. Ivanovsky Federal State Budgetary Institution “NITsEM named after N.F. Gamaleya "of the Ministry of Health of Russia under No. 2931, for the production of live influenza intranasal vaccine for adults and children.